Effects of Different Container Types on (1→3)-ß-D-glucan Recovery.

It has long been known that containers for sample analysis or storage can play a role in endotoxin recovery and have to be taken into account when determining endotoxin concentrations. However, there is little data on the effects of containers regarding (1→3)-ß-D-glucan, which plays a role as a contaminant in endotoxin measurements. To determine the effect of the container on (1→3)-ß-D-glucan measurements, four different types of containers were investigated at different temperatures and stored for up to 28 days. For short-term storage for 3 h at room temperature, no effect of the container on the (1→3)-ß-D-glucan recovery could be observed, but for storage at -20 °C, the results indicate that the storage time and temperature influences (1→3)-ß-D-glucan detection. All containers showed a trend of lower recoveries over time, but the polyethylene container showed a significantly lower recovery compared to the other containers. We also showed that freeze/thaw cycles had a strong influence on the recovery of (1→3)-ß-D-glucan in polyethylene containers. Our study showed that the container can affect not only the detection of endotoxins but also the detection of (1→3)-ß-D-glucans.

Protocol for photoactivation of YAP in cancer cell spheroids embedded in collagen gels.

This protocol describes the necessary preparations and procedures to photo-activate Yes-associated protein (YAP) with optoYAP in cancer cell spheroids in 3D collagen matrices. We detail steps for immunofluorescent staining of the resulting YAP-activated HeLa spheroids. In addition, we describe handling of optoYAP on 2D substrates. While this protocol focuses on the use of optoYAP in 3D HeLa cell culture, it can be modified for other cell types. For complete details on the use and execution of this protocol, please refer to Illes et al. (2021).

Spatio-selective activation of nuclear translocation of YAP with light directs invasion of cancer cell spheroids.

The mechanical properties of the extracellular matrix strongly influence tumor progression and invasion. Yes-associated protein (YAP) has been shown to be a key regulator of this process translating mechanical cues from the extracellular matrix into intracellular signals. Despite its apparent role in tumor progression and metastasis, it is not clear yet, whether YAP activation can actively trigger the onset of invasion. To address this question, we designed a photo-activatable YAP (optoYAP), which allows for spatiotemporal control of its activation. The activation mechanism of optoYAP is based on optically triggered nuclear translocation of the protein. Activation of optoYAP induces downstream signaling for several hours and leads to increased proliferation in two- and three-dimensional cultures. Applied to cancer spheroids, optoYAP activation induces invasion. Site-selective activation of optoYAP in cancer spheroids strikingly directs invasion into the activated direction. Thus, nuclear translocation of YAP may be enough to trigger the onset of invasion.

Biocompatible crosslinked ß-cyclodextrin nanoparticles as multifunctional carriers for cellular delivery.

Nanoparticle-based biomedicine has received enormous attention for theranostic applications, as these systems are expected to overcome several drawbacks of conventional therapy. Herein, effective and controlled drug delivery systems with on-demand release abilities and biocompatible properties are used as a versatile and powerful class of nanocarriers. We report the synthesis of a novel biocompatible and multifunctional material, entirely consisting of covalently crosslinked organic molecules. Specifically, ß-cyclodextrin (CD) precursors were crosslinked with rigid organic linker molecules to obtain small (∼150 nm), thermally stable and highly water-dispersible nanoparticles with an accessible pore system containing ß-CD rings. The nanoparticles can be covalently labeled with dye molecules to allow effective tracking in in vitro cell experiments. Rapid sugar-mediated cell-uptake kinetics were observed with HeLa cells, revealing exceptional particle uptake within only 30 minutes. Additionally, the particles could be loaded with different cargo molecules showing pH-responsive release behavior. Successful nuclei staining with Hoechst 33342 dye and effective cell killing with doxorubicin cargo molecules were demonstrated in live-cell experiments, respectively. This novel nanocarrier concept provides a promising platform for the development of controllable and highly biocompatible theranostic systems.

Liposome-Coated Iron Fumarate Metal-Organic Framework Nanoparticles for Combination Therapy.

One of the main problems for effective treatment of cancer is resistances, which often require combination therapy-for effective treatment. While there are already some potential drug carriers-e.g., liposomes, available for treatment-the effective loading and retention of the desired drug ratio can be challenging. To address this challenge, we propose a new type of drug carrier: liposome-coated metal-organic framework (MOF) nanoparticles. They combine the advantages of liposomes with an easy and efficient loading process. In this work, we present the successful synthesis of liposome-coated MOF nanoparticles via the fusion method. The resulting particles, once loaded, show no premature leakage and an efficient release. Their successful loading with both single and multiple drugs at the same time makes them an interesting candidate for use in combination therapy.